Process for producing tetracycline



United States Patent PROCESS FOR PRODUCING TETRACYCLINE Enzo Zannini,Ermanno Piacenza, and Giuseppe Fabbri, Milan, Italy, assignors toAnkerfarm S.p.A., Milan, Italy, a corporation of Italy N0 Drawing. FiledDec. 9, 1963, Ser. No. 329,222 Claims priority, application Italy, Oct.19, 1963, 21,465/ 63 2 Claims. (Cl. 195-80) ABSTRACT OF THE DISCLOSURE Aprocess for forming tetracycline through the action of the microorganismATCC 15299 on a nutrient medium containing an aqueous extract of Zeamays and strontium carbonate, and having a pH of between 5.7 to 7.0after the medium has been sterilized.

This invention relates to a process for producing tetracycline and moreparticularly to a novel fermentation process for producing tetracyclinein high yield through the use of the microorganism Streptomyces sp.1616.

Streptomyces sp. 1616 was located in the soil of Somma Lombardo, Italy,proximate to Milan, Italy. Streptomyces sp. 1616 differs from anymicroorganism heretofore reported in the literature as being useful forthe production of tetracycline. It produces tetracycline in the presenceof chloride ions. Moreover, We have discovered that the presence ofstrontium carbonate in the fermentation medium markedly improves theyield of tetracycline in fermentation processes employing Streptomycessp. 1616. This likewise has not heretofore been reported in theliterature in connection with any other microorganism.

The principal morphological, physiological, biochemical and culturalcharacteristics of Streptomyces sp. 1616 are as follows:

(1) Vegetative mycelium: Colorless up to twenty-four hours and of abright red color afterwards with different red degrees in conformitywith the different media.

(2) Aerial mycelium: When present it is white, successively grey andbrown.

(3) Spores: When present they are of a greyish-brown color with a drysurface without water condensation.

(4) The growth temperature can be within the range of from 22 to 35 C.,with the optimum being 26-28 C.

Its appearance in the different culture media is as follows:

Glucose-Peptone agar: Reddish brown pigment, red orange vegetativemycelium, aerial mycelium from white to rosy.

Potatoes-plugs: Vegetative mycelium dark brown, aerial mycelium rosy.

Glucose-Asparagine agar: Scanty growth, vegetative mycelium greyishyellow, no aerial mycelium.

Czapek-Dox synthetic agar: No growth produced at all.

Nutrient agar: Scarce growth, sparse brown colonies.

Glucosemeat extract agar: Moderate growth, vegetative mycelium brightbrown.

Agar-potato: Scanty growth, vegetative mycelium reddish-brown, no aerialmycelium.

In Virgilio, Hennegeller, Id Farmaco, Ed. Sc. vol. 3, pp. 164-171 (1960)and in British Patent 775,139 there is described the microorganismStreptomyces psammoticus, which is described as being capable ofproducing tetracycline in the presence of chloride ions. We have made acomparison between the strains Streptomyces sp. 1616 and themorphological and cultural characteristics 3,398,057 Patented Aug. 20,1968 of Streptomyces psammoticus, as described in the literature, and wehave conclusively established that the two strains are different. Thus,a side by side comparison of these organisms reveals the following:

Str. psammoticus Str. sp. 1616 Sporophores 1 Reetus flexibilisRetinaculum apertum. Spore color Olive-buff Red-grey. Sporophores 2Sympodialis-branched Mouopodialis-branched flexuous. open spirals.

Spore color 2 Grisens Cinnamoneus-cinereus:

l Pridham et al., Applied Microbiology, vol. 6, pp. 52-79 (1958).

2 Ettlinger et al., Archiv. Mikrobiologie, vol. 31, pp. 326-358. (1958).

After this first investigation we have compared our strain Streptomycessp. 1616 also with all strains which produce chlortetracycline (andtetracycline in dechlo-rinated media and in the presence of chlorinationinhibitors).

The strains belonging to this group, and the literature referencestherefor are as follows:

Streptomyces aureofaciens described in Duggar, Ann. N.Y. Acad. Sc., vol.51 p. 177 (1948), and US. patent 2,482,055.

Streptomyces viridifaciens described in US. patent 2,712,517.

Streptomyces persimilis described in British patent 799,051.

Streptomyces R0 1441 described in British patent 772,149.

Streptomyces P 4871 described in British patent 790,- 953.

Streptomyces ATCC 11652, 11653, and 11654 described in British Patent787,895.

We have compared Streptomyces aureofaciens ATCC 10762 with Streptomycessp. 1616 by performing the morphological, physiological and culturaltests on the media, which have been used by the different investigatorsfor the classification of Streptomyces aureofaciens; the other strainshave been compared with the data found in the existing literature. Theresults obtained are shown in the following tables:

Peptnnn cm 10 Glucose m 20 NaCl m 5 Agar cm 13 Strain Growth VegetativeAerial Spores mycelium mycelium Str. 1616 Moderate--- Light orange Whiteto rosy- XXX.

to red. white. Aureofaciens Fair Topaze to White to ATCC 10762. orangegreyish yellow white. .ATCC 11652.... XX XX XX XX. ATCC 11653.... XX XXXX XX. ATCC 11654.... XX XX- XX XX. Feoi'aricn XX XX XX XX. R0 1441 XXXX XX XX, P 4871 XX XX XX XX. Persimilis XX XX XX XX. Viridiiaciens...XX XX XX XX. Fuscofaciens.--. XX XX XX XX.

XX-Not described. XXXN0 growth.

GELATINE-DIFCO Str. 1616 Complete liquefaction. Aureofaciens AICC 10762N 0 detectable liquefaction. ATCC 11652 XX. ATCC 11653 XX. ATCC 11654XX. Feofaciens.-.. XX. R0 1441 No liquefaction. P 4871-. Do. Pers1mi11sN0 detectable liquefaction. Viridifaciens.. XX. Fuscofaeiens N oliquefaction.

XXNot described.

GLUOOSE-ASPARAGINE-AGAR Glucose m 10 Asparagine. e 0. 5

KzHPO: cm 0.5

Meat Extract 9m 2 Agar.-. em 18 Distilled H120 to 1, 000 cc.

Strain Growth Vegetative mycclium Aerial mycelium Spores Str. sp. 1616Good Light orange with red dots Rosy grey XXX. Aureofaciens ATCC 10762.Colorless Whi Mouse grey. ATCC 11652 Buff to cinnamon-bull Scarce, whiteWhite to brownish grey to mouse grey. ATCC 11653 Moderate Orange Whiteto light brown. ATCC 11654 Moderate to good Colorless to vim..." XX.Feofaciens Scar Light yellow to light brown Very scarce, light brown todark brown.

Viridifaciens. Good X Light grey Light grey. R0 14 X Yellow tobrownyellow White Grey. P 4871 Good Abundant White to dark grey. Do.Persimilis X Yellow to light brown White to light grey Dark grey.Fuscofaciens Scarce to moderate Purple-brown XXX XXX.

XNot reported by the authors. XXX-No growth.

NUTRIENT AGAR Peptone gm 5 Meat Extractm 5 NaCl gm 5 Agar em 20Distilled H2O to 1,000 cc.

Growth Vegetative mycelium Aerial mycelium Spores Scarce Orange red tobrown-red.-. XXX XXX. Moderate White to greyish White X.

Creamy white... XXX XXX.

Bull colored. Creamy white... XX

X3: Abundant Altnmdant and characteris Viridifaciens. Good X.Fuscofaciens Scarce to moderate- XX." XX. Persimilis do Snow white X.

XNot reported. XXNot described. XXXNo growth.

CZAPEK-DOX SYNTHETIC AGAR Sucrose gm 30 NaNOa 2 FeSOA .g 0. 01

Agan 20 Distilled water to 1,000 cc.

Strain Growth Vegetative mycelium Aerial mycelium Spores Str. sp. 1616No growth N0 growth No growth No growth. Aureoiaciens ATOC 10762.Ahnnd-mt White White to light grey Col. Piping Rock 13A2 to V mousegrey. ATOC 11652.. Scarce XX XXX XX. ATCC 11653--- do XX. White Mousegrey. ATCC 11654 do XXX. Feofaciens or psammoticus X O 41 Scarce Veryscarce Persimilis Moderate to good X Viridifaciens. Scarce XNotreported. XXNot described. XXX-N0 growth. *Maerz A., & Paul M. R, ADictionary of Colors, McGraw-Hill (1950).

GLUCOSE-PEPTONE-AGAR Glueose .gm 30 Peptone. em 10 Agar em 20 Distilledwater to 1, 000 cc. pH=5.9.

Strain Growth Vegetative mycelium Aerial mycelium Spores Str. sp. 1616Moderate Light orange to red White to grey-white to XXX.

grey-rose. Aureofaciens ATCC 10762.. Good Ochre yellow White XXX.

CO 11652 X XX X XX.

ATCC 11654 Feoiaciens or psammotiom XXNot described. XXXN0 growth.

STARCH AGAR Soluble starch um NaNO m 1 m 0.3 m 0.5 gm 1 Agar. ..gmDistilled water to 1,000 cc.

Strain Growth Vegetative mycelium Aerial mycelium Spores Str. sp. 1616Very scarce Colorless XXX XXX. Aureofaciens ATCC 10762... AbundantLifgiht llzrlrvxn to col. Piping White to grey Mouse grey.

0c 2. ATCC 11652 Moderate X. X. Good sporulation white to mouse grey toblack. A'ICC 11653 do White to mouse grey White Light'to dark mousegrey. ATCC 11654 Scarce to moderate White at margins becoming X XXX:

yellowish-grey to brownish-grey. Feotaciens or psammoticus Very scarceColorless O 1441 Very slow and scarce X P 4871." XX. XX Persimilis Veryscarce X X. Viridifaciens XX XX XX XX. Fuscol'aciens Scarce to moderateLight ink blue XXX XXX.

XNot reported. XXNot described. XXXNo growth. 'Maerz A., & Paul M. R., ADictionary of Colors, McGraw-Hill (1950).

AGAR POTATO Potatoes to gm..- 200 Glucosegm.- gm... 20

Vegetative mycelium Aerial mycelium Spores Orange to reddish orangeuuXXX XXX.

Colorless to light grey.. White to grey- XX XX ai ood. Olive-grey andyellow or XXX.

light brown to dark brown.

XNot reported. XX-Not described. XXX-No growth.

X Viridifaciens. R Fuscoiaciens G POTATOES PLUGS Strain GrowthVegetative mycelium Aerial mycelium Spores Str. sp. 1616 Good BrigandRed 2111 White to rosy-grey to ash- Ash-grey.

gTBY. Aureofaciens ATCC 10761.--. Scarce to moderate Yellowish-brown tobrown Greyish white at margins X. ATGC 11652 Good Llght yellow-brown toWhite None to white to browndark olive brown. ish grey. ATCC 11653Moderate Creamy white to creamy XXX XXX.

yellow to mustrad yellow. ATGC 11654 Good Olive brown XXX XXX.

XX XX Feol'aeiens or psammotious. XX

X-Not reported. XX-Not described. XXXNo growth. Maerz A. 6: Paul M. R.,A Dictionary of Colors, McGraw-Hill (1960).

As will be obvious from the above tables and data, tion, the pH of themedium should be between 5.7 and the Str. sp. 1616, owing to itsmorphological, biochemical 6.8. The temperature of the growth medium andthe and cultural characteristics, cannot be identified as befermentationmedium should be bet-ween 24 to longing to any recognized group ofmicro-organisrns de- C., and preferably about 26' to 28 C.

scribed as tetracycline producers. For the growth medium, we have founda preferred We have discovered that the stabilizing action of medium tocontain per 1000 cc.:

strontium carbonate, and mixtures of strontium carbonate Grams andcalcium carbonate, upon the pH, of both the growth Corn dextrin 10-40media and the fermentation media, results in improved Strontiumcarbonate 2-12 growth in the case of the growth media and improved yieldof tetracycline in the case of the fermentation media. Z ea mays fi 18prepared by bolhng 720. grams This is unexpected property, notheretofore reported in Zea mays m 2 ihtefs of Water and then filtenng ithe literature for any comparable microbiological process. mlxmre so asto obtam 'bwwn cPlored filtrate having Furthermore, 'we have discoveredthat maximum yields a PH of about total nitrogen Fonccntmtlon areobtained when the fermentation media comprise either of about and then15 added to a medlum of about fresh or ensiled Zea mays. We have foundthat the Z80! 250 to 750 mays contains growth factors not present inother plants. Ammonium su1fate 1 to 5 grams Thus, when we havesubstituted soya for the Zea mays, W te m the yield of tetracyclinedecreased substantially. The a r m a su Clem quantity to make 1000growth factor contained in Zea mays is capable of be- Beforesterilization the pH should be adjusted to about ing extracted therefromby both acid and alkaline ex- 5.0 with hydrochloric acid. Aftersterilization, the pH traction agents, as well as neutral extractionagents. should be within the range of 6 to 7. The presence of Both thegrowth medium and the fermentation medium the strontium carbonate bothregulates the pH and beneshould be at an initial pH of 6.0 to 7.0.During fermentaficially afiects the yield of tetracycline.

The following examples reveal the growth of Streptomyces sp. 1616, andmay be used to provide an inoculant for the fermentation media:

Example I Spores of the strain Streptomyces sp. 1616 were washed fromthe slant with sterile 1% peptonized water to form a suspensioncontaining about 500,000,000 spores per cc. 1 cc. of this suspension wasused to inoculate a 3 liter flask containing 500 cc. of the followingmedium:

Corn dextrin grams 2O Strontium carbonate do Zea mays filtrate,

prepared as set forth above cc- 250 Ammonium sulfate grams 2 Watersufiicient to make 1000 cc.

The pH of this medium prior to sterilization was adjusted to 5.0 withhydrochloric acid. The pH of the medium after sterilization was 6.35 to6.5.

The growth was continued in the medium for between 2A- and 48 hours.

Example II The procedure of Example I was repeated except that themedium contained:

Corn dextrim "grams-.. 20 Strontium carbonate do.. 10

Zea mays filtrate, prepared as set forth above cc 500 Ammonium sulfate-grams 2 Water sntficient to make 1000 cc.

Example III The procedure of Example I was repeated except that themedium contained:

Corn dextrin gm 20 Strontium carbonate gm.. 10 Zea mays filtrate,prepared as set forth above cc 750 Ammonium sulfate gm 2 Watersufficient to make 1000 cc.

Tetracycline was obtained in high yield when from 2 to 4 weight percentof mycelium, grown between 24 and 48 hours in the nutrient mediadescribed in the aforesaid Examples I, -II and III were inoculated intothe following fermentation media:

Example IV Neutral filtrate of Zea mays cc 250 NI-I Cl gm 4,5 Cornstarch gm 55 Soy-bean gm 10 SrCO gm 8 Spring water to 1000 cc.

1 The neutral filtrate of Zea maps is prepared by boiling 720 grams ofZea maps with 2 liters of water, filtering to produce a liquid of a.brown color having a pH of 6.2, and a. total nitrogen concentration of.02 weight percent.

The pH before the sterilization must be adjusted to 5.0- 5.5 pH afterthe sterilization-6.2.

Example V Neutral filtrate of Zea mays cc 500 NI-I C1 gm 4,5 Corn starchgm 55 Soy-bean gm 10 SrCO gm 8 Spring water to 1000 cc.

See footnote 1 of Example IV.

The pH before the sterilization must be adjusted to 5.05.5 pI-I afterthe sterilization6.2

Spring water to 1000 cc.

1 The Alkaline filtrate of Zeamays is prepared by suspending 720 gramsof Zea ma/ys in '2 liters of water, and ad usting the pH to 9.0 with 20weight percent sodium. hydroxide. The mixture is then brought itoboiling for 30 minutes and filtered. The filtrate is a brown-yellowishliquid having a pH of 1:90 with a total nitrogen concentration of .022weight percen The pH before the sterilization must be adjusted to5.0-5.5 pH after the sterilization6.2

Example IX Alkaline filtrate of Zea mays 1 cc 750 NH Cl gm 4,5 Cornstarch gm Soy-bean gn1 10 SrCO gm 8 Spring water to 1000 cc.

1 See footnote 1 of Example VII.

The pH before the sterilization must be adjusted to 5.0-5.5 pH after thesterilization-6.2

Example X Acid filtrate of Zea mLWS 1 cc 250 NH CI gm 4,5 Corn starch gm55 Soy-bean gm l0 SrCO gm 8 Spring water to 1000 cc.

The acid filtrate of Zea maps is prepared by suspending 720 grams of Zea'mays in 2 liters of water, acidifying the mixture with 20 vol. percentof hydrochloric acid to bring it to a. pH of 3, boiling and thenfiltering the mixture to yield a yelliiwish liquid having a nitrogencontent of .02 Weight percen The pH before the sterilization must beadjusted to 5.0-5.5 pH after the sterilization-6.2.

Example XI Acid filtrate of Zea mays 1 cc 500 NH Cl g 4.5 Corn Starch g55 Soy-bean g 10 SrC-O g 8 Spring water to 1000 cc.

1 See footnote 1 of Example X. The pH before the sterilization must beadjusted to 5.0-5.5 pH after the sterilization-6.2.

Example XII Acid filtrate of Zea mays 1 cc 750 NH Cl g 4.5 Corn Starch g55 Soy-bean g 10 SrCO g 8 Spring water to 1000 cc.

The acid filtrate o-f Zea mays is prepared by suspending 720 grams ofZea maps in -2 liters of water, acidifying the mixture with 20 Wt.percent of hydrochloric llcl tl to bring it to a. pH of 3, boiling andthen filtering the mixture to yield a yellipwish liquid having anitrogen content of 0.2 weight percen The pH before the sterilizationmust be adjusted to 5.0-5.5 pH after the sterilization6.2

We have found that very high yields of tetracycline may be obtained fromthe aforesaid fermentation media. The tetracycline may be recovered fromthe fermentation media by the recovery method set forth in the copendingapplication Ser. No. 329,298 and now US. Patent No. 3,272,862 filed oneven date herewith in the names of Ezio Caputo, Giovanni Bonfanti, andEnzo Zannini, disclosure of which is incorporated herein by reference.However, other known methods for recovering tetracycline from afermentation medium may be used, and form no part of the presentinvention.

We have determined that the highest yields of tetracycline are obtainedwhen strontium carbonate is employed as the 'pH regulator for thefermentation medium. However, other alkaline earth metal carbonates,such as ban'um carbonate and calcium carbonate may be used, preferablyin conjunction with strontium carbonate. If used in place of thestrontium carbonate, the yield of tetracycline is reduced.

Furthermore, as above-indicated, the yield of tetracycline is markedlyreduced if the Zea mays constituent is replaced by soy-bean or othernutrient. We believe that there is a growth factor present in Zea mayswhich materially increases the yield of tetracycline when theStreptomyces sp. 1616 is employed as the microorganism. However, we havesuccessfully recovered tetracycline from nutrient media containing noZea mays but other nutrients, such as soy-bean. When Streptomyces sp.1616 is employed, it is not necessary to remove chloride ions from thenutrient medium.

A sample of the spores of Streptomyces sp. 1616 have been deposited withthe American Type Culture Collection, Washington, DC, and has beendesignated ATCC 15299.

The present invention may be embodied in other specific forms withoutdeparting from the spirit or essential attributes thereof and,accordingly, reference should be made to the appended claims, ratherthan to the foregoing specification as indicating the scope of theinvention.

We claim:

1. A process for producing tetracycline which comprises producingtetracycline by microbiological fermentation through the action of themicroorganism Streptomyces ATCC 15299 on a nutrient medium containing anaqueous extract of Zea mays and strontium carbonate, with such nutrientmedium having a pH of between 5.7 to 7.0 after such medium has beensterilized, and recovering said tetracycline from said nutrient medium.

2. A process in accordance with claim 1 in which the nutrient mediumcontains chloride ions.

References Cited UNITED STATES PATENTS 2,678,903 5/1954 G-apen et a1.19536 3,037,916 6/1962 Goodman l80 FOREIGN PATENTS 201,870 5/1956Australia 80 MAURICE W. GREENSTEIN, Primary Examiner.

